The research projects in my group, as outlined below, are centered on an analysis of the factors that regulate chondrogenesis and cartilage development. 1. Analysis of the structure and function of the COMP Gene. Cartilage oligomeric matrix protein (COMP) is a specific component of cartilage, tendon and ligament. Mutations within the COMP gene lead to skeletal malformations and early onset arthritis. While COMP appears to play a crucial role in cartilage biology its exact role is unknown. Here we analyze the role of COMP in regulating chondrogenesis and osteogenesis. We have utilized both human and mouse mesenchymal stem cells (MSCs) since these cells can differentiate in vitro into chondrocytes and osteoblasts. We have ectopically expressed COMP (wild-type and mutants) via plasmid and retroviral expression vectors and have assayed the resulting effects on chondrogenesis and osteogenesis. We find that COMP inhibits osteogenesis while enhancing chondrogenesis in vitro. By expressing specific domains of COMP in these human MSCs we will be able to isolate and identify regions of COMP that specify its chondro-enhancing and osteo-inhibiting functions. We have also assessed the role of COMP on cell proliferation and have found that specific domains of COMP have growth inhibitory functions. Interestingly, these domains appear to be effective at blocking the proliferation of cancer cells. Future analysis will be directed at identifying the cellular receptors for COMP and determining its mode of action in terms of differentiation and proliferation. In addition, we will generate COMP transgenic mice. We will express wild-type and mutant COMP proteins in cartilage to determine the extent of any developmental defects in these mice and in particular those that affect cartilage. These approaches should help to shed light on the contribution of this important ECM molecule to cartilage biology and osteoarthritis. 2. Cell Cycle Regulators of Chondrogenesisand Cell Survival. The goal of these studies is to better understand the link between cell cycle control and chondrogenesis, to determine how specific cell cycle regulators play dual roles by controlling both proliferation and differentiation. We have focused on a group of cell cycle inhibitory proteins; namely the p21family of cyclin dependent kinase inhibitors. Since p21 is a growth inhibitor, its levels are usually low in growing cells. However we find that p21 levels are very high in growing human MSCs and chondrocytes. This is especially relevant with regard to cartilage biology considering the recent findings that p21 blocks apoptosis in mammalian cells. This is consistent with our findings that p21 is upregulated during chondrogenesis, under conditions favorable for apoptosis. The fact that apoptosis is blocked suggests that p21 aids in the survival of chondrocytes under adverse conditions. These data indicate that p21 would play a key role in chondrogenesis in addition to its role in the cell cycle. Using standard gene transfer approaches we have expressed p21 in cells have found it to under a unique form of post-translational modification. Our future experiments will be directed toward understanding the form of this modification. Additionally, by overexpressing p21 and by lowering its levels by RNAi experiments, we will determine how p21 affects chondrogenesis and cell survival in vitro. These experiment will provide important insight into the mechanism by which a key cell cycle regulator also plays an essential role in cell survival and differentiation. 3. Genetic Screens. It is well recognized that there are multiple causes for the initiation of OA, some of which involve the effect of toxic factors directly on cartilage. In an attempt to identify genes that play critical roles in chondrocyte survival and chondrogenesis, we have initiated functional screens, using human cDNA libraries cloned into a retroviral expression vector. These retroviral cDNA libraries were used to infect chondrosarcoma cells, MSCs and chondrocytes. In our first screens we then have begun the process of selection for specific characteristics, such as for survival of cells treated with agents known to exert toxic effects on bone and cartilage. For example, we have assayed for proliferation in media lacking growth factors or in the presence of toxic agents such as retinoic acid (RA), tumor necrosis factor (TNF), interleukin 1 (IL1) or the heavy metals lead (Pb) and gadolinium (Gd). Through this approach we hope to identify novel factors that aid in the survival of these cells. The results of these initial screens is very promising for it has helped us identify a number of genes that one would not normally expect to be part of these processes. In the future we will use the information derived from these screens along with retroviral gene transfer approaches to express these potential chondro-protective genes in both cartilage and in MSCs. We will then determine the outcome of this expression on the survivability of chondrocytes in vitro and cartilage in vivo.